實驗室手扎

STEP 1. Isolate mouse DRG and place into 15 ml DMEM (serum-free) solution. Centrifuge using 800rpm, at room temperature, for 2 minutes and then remove supernatant.

STEP 2. Add 1 ml DMEM (serum-free) containing 0.125% Type Ia Collagenase (Stock=2.5%, take 50 ul into 950 ul DMEM), and incubate in 37 0C for 1.5 hours.

STEP 3. Add 5 ml DMEM, and then gentled mix for a while. Centrifuge at 800rpm, RT for 2 minutes and then remove supernatant.

STEP 4. Incubate DRG in 1 ml 0.25% trypsin (EDTA-free) at 370C for 15 minutes. Add 5 ml DMEM (with serum) and mix to inactivate enzymatic activity of trypsin. Spin down (800rpm) and remove supernatant. Wash with 10 ml DMEM (serum-free), spin down, and then remove supernatant.

STEP 5. Add 2 ml DMEM (serum-free) and triturate DRG with flame polished Pasteur pipette for several( at least 7) times. Rinse the glass pipette with DMEM before contacting the DRG (This procedure will prevent adhesion of DRG on the glass wall).

STEP 6. Transfer to a new tube containing 15 ml DMEM and leave undisrupted for 3 minutes. Fiber debris will fall first in the bottom of the tube. Collect the top 11 ml medium into a new tube without disturbing the debris below. Spin down the DRG at 1000 rpm for 5 minutes.

STEP 7. Resuspend pellet in 2 ml DMEM (containing 1% P/S, 10% FCS, and 2 mM glutamine) and then seed in 3.5 cm dish (coated with 0.1 mg/ml Poly L-lysine).